DIPLOMARBEIT Titel der Diplomarbeit „Construction of an ORF34 deletion φCh1 strain and secretion of putative tail fibre proteins“ Verfasserin Dimmel Katharina angestrebter akademischer Grad Magistra der Naturwissenschaften (Mag.rer.nat.) Wien, 2013 Studienkennzahl lt. Studienblatt: A 441 Studienrichtung lt. Studienblatt: Diplomstudium Genetik – Mikrobiologie (Stzw) UniStG Betreut von: Ao. Univ.-Prof. Dipl.-Biol. Dr. Angela Witte 2 3 4 Table of the contents 1. INTRODUCTION ........................................................................................................................... 10 1.1 ARCHAEA – THE THIRD DOMAIN OF LIFE ....................................................................................................10 1.1.1 Diversity and abundance ................................................................................................................. 11 1.1.2 Archaea a mosaic of Bacteria and Eukarya ..................................................................................... 13 1.1.3 Archaea, Bacteria and Eukarya in contrast ..................................................................................... 13 1.1.3.1 Structural aspect .....................................................................................................................................13 1.1.3.2 Genetic aspect .........................................................................................................................................14 1.1.3.3 Replication process .................................................................................................................................14 1.1.3.4 Transcription ...........................................................................................................................................16 1.1.3.5 Translation ..............................................................................................................................................16 1.1.4 Life in extreme environments .......................................................................................................... 17 1.1.4.1 Physically extremes .................................................................................................................................18 1.1.4.2 Geochemically extremes: ........................................................................................................................19 1.1.5 Halo-alkaliphilic Archaea and their adaption to extreme environments ......................................... 20 1.1.5.1 Cell envelope and osmotic adaptation ....................................................................................................21 1.1.5.2 Haloalkaliphilic adaptation of proteins ...................................................................................................22 1.1.5.3 Adaptation of protein secretion systems of haloalkaliphilic organisms ..................................................23 1.1.5.4 Metabolic aspects of alkaliphilic Haloarchaea ........................................................................................25 1.1.6 Biotechnical applications of (haloalkaliphilic) Archaea ................................................................... 26 1.2 NATRIALBA MAGDII .............................................................................................................................27 1.2.1 General characteristics of Natrialba magadii .................................................................................. 27 1.2.2 Laboratory strains of Natrialba magadii ......................................................................................... 27 1.2.3 Working with Natrialba magadii ..................................................................................................... 28 1.2.3.1 Transformation of Natrialba magadii .....................................................................................................28 1.2.3.2 Vectors and genetic markers for Natrialba magadii ...............................................................................29 1.2.3.3 Natrialba extracellular protease (Nep) ....................................................................................................30 1.3 ARCHAEAL VIRUSES .............................................................................................................................31 1.3.1 The halophage φCh1 infecting Natrialba magadii .......................................................................... 32 5 1.3.2 Morphology and structural components of φCh1 ........................................................................... 32 1.3.3 Life cycle of the temperate φCh1 ..................................................................................................... 34 1.3.4 Genomic organization of φCh1 ........................................................................................................ 35 1.3.5 The invertible region of the φCh1 genome ...................................................................................... 37 1.3.6 Aspects of the putative tail fibre proteins ........................................................................................ 39 1.3.7 Virus – host interaction .................................................................................................................... 41 2. MATERIALS AND METHODS ......................................................................................................... 45 2.1 MATERIALS: ......................................................................................................................................45 2.1.1 Strains .............................................................................................................................................. 45 2.1.2 Growth media .................................................................................................................................. 45 2.1.3 Additives .......................................................................................................................................... 47 2.1.4 Vectors ............................................................................................................................................. 47 2.1.5 Primer .............................................................................................................................................. 49 2.1.6 DNA and protein markers ................................................................................................................ 50 2.1.6.1 DNA ladders ............................................................................................................................................50 2.1.6.2 Protein ladders ........................................................................................................................................51 2.1.7 Enzymes and corresponding buffers ................................................................................................ 51 2.1.7.1 Restriction ...............................................................................................................................................51 2.1.7.2 PCR ..........................................................................................................................................................51 2.1.7.3 Other enzymes ........................................................................................................................................52 2.1.8 Antibodies ........................................................................................................................................ 52 2.1.8.1 Primary antibodies ..................................................................................................................................52 2.1.8.2 Secondary antibodies ..............................................................................................................................52 2.1.9 KITs .................................................................................................................................................. 53 2.1.10 Solutions and reagents for DNA methods ........................................................................................ 53 2.1.10.1 Gelelectrophoresis ..................................................................................................................................53 2.1.10.2 Gel extraction from polyacrylamide gels .................................................................................................54 2.1.10.3 Southern blot analysis .............................................................................................................................54 2.1.11 Solutions and reagents for proteins methods .................................................................................. 55 2.1.11.1 SDS-PAGE and western blot analysis .......................................................................................................55 6 2.1.11.2 Protein purification under denaturing conditions (E.coli) .......................................................................56 2.1.12 Isolation of chromosomal DNA from Nab. magadii ......................................................................... 57 2.1.12.1 Extraction of plasmid DNA from Nab. magadii .......................................................................................57 2.1.12.2 Buffers and solution for phage methods .................................................................................................57